1,3-butadiene (BD) is a known rodent and human carcinogen. However, the DNA adducts responsible for base pair mutations have not been identified. We hypothesize that N1- (hydroxybutene)-deoxyinosine (N1-HB-dI) and N1-trihydroxybutane-deoxyinosine (N1-THB-dl) adducts are responsible of the mutations at A:T base pairs. This hypothesis is based on studies showing that N1-HB-dl adducts are highly mutagenic in site directed mutation studies in E. coli and COS cells. There is no information on the formation, persistence or repair of these adducts in vitro or in vivo. Therefore, in the proposed studies we will synthesize the N1-HB-dl and N1-THB-dl adduct standards and develop a method for their detection and analysis by LC-ESI-MS. The developed methods will then be applied to DNA samples from human TK6 cells, exposed to 1,2-epoxy-3-butene (EB) or 1,2;3,4-diepoxybutane (DFB). Additionally, these methods will be used to analyze N1-inosine adducts in mice and rats exposed to different concentrations of BD, EB or DEB to evaluate the dosimetry of adduct formation in vivo. Further, tissues from mice and rats treated with BD for 10 days and sacrificed 2h, 1, 3 or 6 days post exposure will be analyzed to calculate the adduct half-lives and give insight on the rate of repair. Finally, the number of N1-inosine adducts will be compared with previously analyzed amounts of N7-gunaine adducts (N7-HB-gunaine and N7-THB-gunine) and the hprt mutations in mice and in TK6 cells. Addressing this hypothesis is essential for determining the sources of BD mutations and will ultimately increase our ability to accurately assess the risk of BD to humans.